Delivery of cell-killing doses of ionizing radiation to tumors is the objective of antibody-directed radioimmunotherapy (RIT). In practice, however, collateral damage to healthy bone marrow and kidneys limits the maximum delivered radiation dose. Pretargeted RIT (PRIT) aims to overcome this limitation by separating the pharmacokinetics of tumor targeting and radionuclide delivery. The overarching perspective of this resubmitted renewal proposal is that the principle of PRIT is sound, but that to reach its full potential the protein targeting agents must be optimized, and the pharmacokinetics of tumor penetration must be subjected to rigorous engineering analysis. This project brings together faculty from Biological Engineering and Nuclear Medicine to collaboratively develop essential reagents and dosing strategies to enable PRIT to be maximally effective. In the previous project period, bispecific antibodies (bsAbs) were engineered with picomolar affinity both to chelated metal ions and to tumor antigens (specifically, the colorectal carcinoma antigens A33 or CEA). New data is presented that demonstrates the robust expression and solubility characteristics of the bsAbs. Biodistribution studies in mouse xenograft models helped establish the parameters for the proposed optimization of pretargeting dosing strategies. Theoretical analyses have provided predictions of the balances amongst antibody binding, diffusion, endocytic uptake, capillary extravasation, and systemic clearance that together determine how far antibodies reach into tumors. This essentially complete tumor microdistribution theory is not limited to PRIT, but is salient for all antibody-based therapeutics. In the next project period, PRIT protocols will be optimized in tumor-xenografted mice, guided by mathematical modeling. Key variables are tumor antigen choice (A33 vs. CEA); bolus dose of the bispecific antibody; bolus dose of a blood pool blocking/clearing agent; waiting time for administration of the clearing agent and subsequent radiometal chelate administration; and bolus dose size of the radiometal chelate. Further improvements in antibody tumor uptake will be pursued by protein engineering methods. A new aim has been added to evaluate toxicity and anti-tumor efficacy in tumor-xenografted mice. By emphasizing principles over ad hoc empirical tinkering, these studies are aimed at establishing a firm scientific foundation from which to develop PRIT. The approaches thus developed should be more readily generalizable. The project has strong momentum and talented graduate students fully engaged in work on each of the Specific Aims.